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Abstract:

The present invention is concerned with the provision of means and
methods for gene expression. Specifically, it relates to a polynucleotide
comprising an expression control sequence which allows for bidirectional
expression of two nucleic acid of interest being operation linked thereto
in opposite orientations. Furthermore, vectors, host cells, non-human
transgenic organisms and methods for expressing nucleic acids of interest
are provided which are based on the said polynucleotide.

Claims:

1. A polynucleotide comprising an expression control sequence which
allows for bidirectional expression of two nucleic acids of interest
being operatively linked thereto in opposite orientations, wherein said
expression control sequence being is selected from the group consisting
of: (a) an expression control sequence having a nucleic acid sequence as
shown in any one of SEQ ID NOs: 1 to 3; (b) an expression control
sequence having a nucleic acid sequence which is at least 80% identical
to a nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3; (c) an
expression control sequence having a nucleic acid sequence which
hybridizes under stringent conditions to a nucleic acid sequence as shown
in any one of SEQ ID NOs: 1 to 3; (d) an expression control sequence
having a nucleic acid sequence which hybridizes to a nucleic acid
sequence located upstream of an open reading frame sequence shown in SEQ
ID NO: 4; (e) an expression control sequence having a nucleic acid
sequence which hybridizes to a nucleic acid sequence located upstream of
an open reading frame sequence encoding an amino acid sequence as shown
in SEQ ID NO: 5; (f) an expression control sequence having a nucleic acid
sequence which hybridizes to a nucleic acid sequence located upstream of
an open reading frame sequence being at least 80% identical to an open
reading frame sequence as shown in SEQ ID NO: 4, wherein the open reading
frame encodes a 60S acidic ribosomal protein P3; (g) an expression
control sequence having a nucleic acid sequence which hybridizes to a
nucleic acid sequence located upstream of an open reading frame encoding
an amino acid sequence being at least 80% identical to an amino acid
sequence as shown in SEQ ID NO: 5, wherein the open reading frame encodes
a 60S acidic ribosomal protein P3; (h) an expression control sequence
obtainable by 5' genome walking or by thermal asymmetric interlaced
polymerase chain reaction (TAIL-PCR) on genomic DNA from the first exon
of an open reading frame sequence as shown in SEQ ID NO: 4; (i) an
expression control sequence obtainable by 5' genome walking or TAIL PCR
on genomic DNA from the first exon of an open reading frame sequence
being at least 80% identical to an open reading frame as shown in SEQ ID
NO: 4, wherein the open reading frame encodes a 60S acidic ribosomal
protein P3; and (j) an expression control sequence obtainable by 5'
genome walking or TAIL PCR on genomic DNA from the first exon of an open
reading frame sequence encoding an amino acid sequence being at least 80%
identical to an amino acid sequence encoded by an open reading frame as
shown in SEQ ID NO: 5, wherein the open reading frame encodes a 60S
acidic ribosomal protein P3.

2. The polynucleotide of claim 1, wherein said polynucleotide further
comprises at least one nucleic acid of interest being operatively linked
to the expression control sequence.

3. The polynucleotide of claim 1 , wherein said polynucleotide further
comprises at least one nucleic acid of interest being operatively linked
to the expression control sequence in each of the opposite orientations.

4. The polynucleotide of claim 1, wherein said nucleic acid of interest
is heterologous with respect to the expression control sequence.

5. A vector comprising the polynucleotide of claim 1.

6. The vector of claim 5, wherein said vector is an expression vector.

7. A host cell comprising the polynucleotide of claim 1 or a vector
comprising said polynucleotide.

8. The host cell of claim 7, wherein said host cell is a plant cell.

9. A non-human transgenic organism comprising the polynucleotide of claim
1 or a vector comprising said polynucleotide.

10. The non-human transgenic organism of claim 9, wherein said organism
is a plant or a plant seed.

11. A method for expressing a nucleic acid of interest in a host cell
comprising: (a) introducing the polynucleotide of claim 1 or a vector
comprising said polynucleotide into a host cell; and (b) expressing at
least one nucleic acid of interest in said non-human transgenic organism.

12. The method of claim 11, wherein said host cell is a plant cell.

13. A method for expressing a nucleic acid of interest in a non-human
organism comprising: (a) introducing the polynucleotide of claim 1 or a
vector comprising said polynucleotide into the non-human organism; and
(b) expressing at least one nucleic acid of interest in said non-human
transgenic organism.

14. The method of claim 13, wherein said non-human transgenic organism is
a plant or seed thereof.

15. The method of claim 13, wherein said at least one nucleic acid of
interest is expressed in each orientation from the expression control
sequence.

16. (canceled)

Description:

[0001] The present invention is concerned with the provision of means and
methods for gene expression. Specifically, it relates to a polynucleotide
comprising an expression control sequence which allows for bidirectional
expression of two nucleic acid of interest being operatively linked
thereto in opposite orientations. Furthermore, vectors, host cells,
non-human transgenic organisms and methods for expressing nucleic acids
of interest are provided which are based on the said polynucleotide.

[0002] The production of transgenic plants is a fundamental technique of
plant biotechnology and, thus, an indispensible prerequisite for
fundamental research on plants, and for producing plants having improved,
novel properties for agriculture, for increasing the quality of human
foods or for producing particular chemicals or pharmaceuticals. A basic
prerequisite for transgenic expression of particular genes in plants is
the provision of plant-specific promoters. Various plant promoters are
known. The constitutive promoters which are currently predominantly used
in plants are almost exclusively viral promoters or promoters isolated
from Agrobacterium such as, for example, the cauliflower mosaic virus
promoter CaMV355 (Odell et al. (1985) Nature 313:810-812). The increasing
complexity of the work in plant biotechnology often requires
transformation with a plurality of expression constructs. Multiple use of
one and the same promoter is problematic especially in plants, because
the multiple presence of identical regulatory sequences may result in
gene activity being switched off (silencing) (Kumpatla et al. (1998) TIBS
3:97-104; Selker (1999) Cell 97:157-160). There is thus an increasing
need for novel promoters. An alternative way of dealing with this problem
is the use of so-called "bidirectional" promoters, i.e. regulatory
sequences which result in transcription of the upstream and downstream
DNA sequences in both direction. It is possible in this case for example
for target gene and marker gene to be introduced into a cell under the
control of one DNA sequence.

[0003] Transgenic expression under the control of bidirectional promoters
has scarcely been described to date. The production of bidirectional
promoters from polar promoters for expression of nucleic acids in plants
by means of fusion with further transcriptional elements has been
described (Xie M (2001) Nature Biotech 19: 677-679). The 35S promoter has
likewise been converted into a bidirectional promoter (Dong J Z et al.
(1991) BIO/TECHNOLOGY 9: 858-863). WO 02/64804 describes the construction
of a bidirectional promoter complex based on fusion of enhancer and
nuclear promoter elements of various viral (CaMV 35S, CsVMV) and plant
(Act2, PRb1b) sequences. US20020108142 describes a regulatory sequence
from an intron of the phosphatidy-linositol transfer-like protein IV from
Lotus japonicus

[0004] (PLP-IV; GenBank Acc. No.: AF367434) and the use thereof as
bidirectional promoter. This intron fragment has a transcriptional
activity only in the infection zone of the nodules. Other tissues, roots,
leaves or flowers show no stain. Plant promoters permitting
bidirectional, ubiquitous (i.e. substantially tissue-nonspecific) and
constitutive expression in plants have not been disclosed to date. WO
03/006660 describes a promoter of a putative ferredoxin gene, and
expression constructs, vectors and transgenic plants comprising this
promoter. The isolated 836 by 5'-flanking sequence fused to the
glucuronidase gene surprisingly show a constitutive expression pattern in
transgenic tobacco. The sequence corresponds to a sequence segment on
chromosome 4 of Arabidopsis thaliana as deposited in GenBank under the
Acc. No. Z97337 (version Z97337.2; base pair 85117 to 85952; the gene
starting at by 85953 is annotated with strong similarity to ferredoxin
[2Fe-2S] I, Nostoc muscorum"). The activity detectable in the
anthers/pollen of the closed flower buds was only weak, and in mature
flowers was zero. Contrary to the prejudice derived from the literature
findings against suitability of the promoter for efficient expression of
selection markers (for example based on the presumed leaf specificity or
the function in photosynthetic electron transport), it was possible to
demonstrate highly efficient selection by combination with, for example,
the kanamycin resistant gene (nptll). WO 03/006660 describes merely the
use as "normal" constitutive promoter. Use as bidirectional promoter is
not disclosed. In order to integrate a maximum number of genes into a
plant genome via a transfer complex, it is necessary to limit the number
and size of regulatory sequences for expressing transgenic nucleic acids.
Promoters acting bidirectionally contribute to achieving this object. It
is particularly advantageous to use a bidirectional promoter when its
activities are present coordinated in the same strength and are located
on a short DNA fragment. Since there is little acceptance for the use of
viral sequences for expression in transgenic plants, it is advantageous
to use regulatory sequences which are likewise from plants. WO2005/019459
describes a bidirectional promoter from Arabidopsis thaliana which allows
for bidirectional expression in various tissues in transgenic tobacco or
canola plants.

[0005] However, there is a clear need for bidirectional expression of
transgenes in a timely restriced or tissue specific manner. Specifically,
bidirectional expression systems allow for controlling expression of
transgenes in a stoichiometric manner. Moreover, the number of expression
cassettes to be introduced into an organism for heterologous gene
expression can be reduced since in a bidirectional expression system, one
expression control sequence governs the expression of two nucleic acids
of interest.

[0006] Thus, the technical problem underlying this invention may be seen
as the provision of means and methods which allow for complying with the
aforementioned needs. The technical problem is solved by the embodiments
characterized in the claims and herein below.

[0007] Accordingly, the present invention relates to a polynucleotide
comprising an expression control sequence which, preferably, allows for
bidirectional expression of two nucleic acid of interest being
operatively linked thereto in opposite orientations, said expres- sion
control sequence being selected from the group consisting of: [0008]
(a) an expression control sequence having a nucleic acid sequence as
shown in any one of SEQ ID NOs: 1 to 3; [0009] (b) an expression control
sequence having a nucleic acid sequence which is at least 80% identical
to a nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 3; [0010]
(c) an expression control sequence having a nucleic acid sequence which
hybridizes under stringent conditions to a nucleic acid sequence as shown
in any one of SEQ ID NOs: 1 to 3; [0011] (d) an expression control
sequence having a nucleic acid sequence which hybridizes to a nucleic
acid sequences located upstream of an open reading frame sequence shown
in SEQ ID NO: 4; [0012] (e) an expression control sequence having a
nucleic acid sequence which hybridizes to a nucleic acid sequences
located upstream of an open reading frame sequence encoding an amino acid
sequence as shown in SEQ ID NO: 5; [0013] (f) an expression control
sequence having a nucleic acid sequence which hybridizes to a nucleic
acid sequences located upstream of an open reading frame sequence being
at least 80% identical to an open reading frame sequence as shown in SEQ
ID NO: 4, wherein the open reading frame encodes a 60S acidic ribosomal
protein P3; [0014] (g) an expression control sequence having a nucleic
acid sequence which hybridizes to a nucleic acid sequences located
upstream of an open reading frame encoding an amino acid sequence being
at least 80% identical to an amino acid sequence as shown in SEQ ID NO:
5, wherein the open reading frame encodes a 60S acidic ribosomal protein
P3; [0015] (h) an expression control sequence obtainable by 5' genome
walking or by thermal asymmetric interlaced polymerase chain reaction
(TAIL-PCR) on genomic DNA from the first exon of an open reading frame
sequence as shown in SEQ ID NO: 4; and [0016] (i) an expression control
sequence obtainable by 5' genome walking or TAIL PCR on genomic DNA from
the first exon of an open reading frame sequence being at least 80%
identical to an open reading frame as shown in SEQ ID NO: 4, wherein the
open reading frame encodes a 60S acidic ribosomal protein P3; and [0017]
(j) an expression control sequence obtainable by 5' genome walking or
TAIL PCR on genomic DNA from the first exon of an open reading frame
sequence encoding an amino acid sequence being at least 80% identical to
an amino acid sequence encoded by an open reading frame as shown in SEQ
ID NO: 5, wherein the open reading frame encodes a 60S acidic ribosomal
protein P3.

[0018] The term "polynucleotide" as used herein refers to a linear or
circular nucleic acid molecule. It encompasses DNA as well as RNA
molecules. The polynucleotide of the present invention is characterized
in that it shall comprise an expression control sequence as defined
elsewhere in this specification. In addition to the expression control
sequence, the polynucleotide of the present invention, preferably,
further comprises at least one nucleic acid of interest being operatively
linked to the expression control sequence and/or at least one a
termination sequence or transcription. Thus, the polynucleotide of the
present invention, preferably, comprises an expression cassette for the
expression of at least one nucleic acid of interest. More preferably, the
polynucleotide comprises at least one expression cassette comprising a
nucleic acid of interest and/or a terminator sequence in each
orientation, i.e. the expression control sequence will be operatively
linked at . Said expression cassettes are, more preferably, operatively
linked to the expression both ends to at least one expression cassette,
the transcription of which is governed by the said expression control
sequence in opposite orientations, i.e. from one DNA strand in one
direction and from the other DNA strand in the opposite direction. It
will e understood that the polynucleotide, also preferably, can comprise
more than one expression cassettes for each direction. Therefore,
polynucleotides comprising expression cassettes with at least two, three,
four or five or even more expression cassettes for nucleic acids of
interest are also contemplated by the present invention. Furthermore, it
will e not necessary to have equal numbers of expression cassettes for
each of the two orientations, e.g., one direction may comprise two
expression cassettes while the other direction of transcription from the
expression control sequence may comprise only one expression cassette.

[0019] Instead of a nucleic acid of interest, the at least one expression
cassette can also comprise a multiple cloning site and/or a termination
sequence for transcription. In such a case, the multiple cloning site is,
preferably, arranged in a manner as to allow for operative linkage of a
nucleic acid to be introduced in the multiple cloning site with the
expression control sequence. In addition to the aforementioned
components, the polynucleotide of the present invention, preferably,
could comprise components required for homologous recombination, i.e.
flanking genomic sequences from a target locus. However, also
contemplated is a polynucleotide which essentially consists of the said
expression control sequence.

[0020] The term "expression control sequence" as used herein refers to a
nucleic acid which is capable of governing the expression of another
nucleic acid operatively linked thereto, e.g. a nucleic acid of interest
referred to elsewhere in this specification in detail. An expression
control sequence as referred to in accordance with the present invention,
preferably, comprises sequence motifs which are recognized and bound by
polypeptides, i.e. transcription factors. The said transcription factors
shall upon binding recruit RNA polymerases, preferably, RNA polymerase I,
II or III, more preferably, RNA polymerase II or III, and most
preferably, RNA polymerase II. Thereby the expression of a nucleic acid
operatively linked to the expression control sequence will be initiated.
It is to be understood that dependent on the type of nucleic acid to be
expressed, i.e. the nucleic acid of interest, expression as meant herein
may comprise transcription of RNA polynucleotides from the nucleic acid
sequence (as suitable for, e.g., anti-sense approaches or RNAi
approaches) or may comprises transcription of RNA polynucleotides
followed by translation of the said RNA polynucleotides into polypeptides
(as suitable for, e.g., gene expression and recombinant polypeptide
production approaches). In order to govern expression of a nucleic acid,
the expression control sequence may be located immediately adjacent to
the nucleic acid to be expressed, i.e. physically linked to the said
nucleic acid at its 5''end. Alternatively, it may be located in physical
proximity. In the latter case, however, the sequence must be located so
as to allow functional interaction with the nucleic acid to be expressed.
An expression control sequence referred to herein, preferably, comprises
between 200 and 5,000 nucleotides in length. More preferably, it
comprises between 500 and 2,500 nucleotides and, more preferably, at
least 1,000 nucleotides. As mentioned before, an expression control
sequence, preferably, comprises a plurality of sequence motifs which are
required for transcription factor binding or for conferring a certain
structure to the polynucletide comprising the expression control
sequence. Sequence motifs are also sometimes referred to as
cis-regulatory elements and, as meant herein, include promoter elements
as well as enhancer elements. The expression control sequence of the
present invention allows for bidirectional expression and, thus,
comprises cis-regulatory elements which can recruit RNA polymerases at
two different sites and release them in opposite directions as to enable
bidirectional transcription of nucleic acids operatively linked to the
said expression control sequence. Thus, one expression control sequence
will be sufficient to drive transcription of two nucleic acids
operatively linked thereto. Preferred expression control sequences to be
included into a polynucleotide of the present invention have a nucleic
acid sequence as shown in any one of SEQ ID NOs: 1 to 3.

[0021] Further preferably, an expression control sequence comprised by a
polynucleotide of the present invention has a nucleic acid sequence which
hybridizes to a nucleic acid sequences located upstream of an open
reading frame sequence shown in any one of SEQ ID NO: 4, i.e. is a
variant expression control sequence. It will be understood that
expression control sequences may slightly differ in its sequences due to
allelic variations. Accordingly, the present invention also contemplates
an expression control sequence which can be derived from an expression
control sequence as shown in any one of SEQ ID NOs: 1 to 3. Said
expression control sequences are capable of hybridizing, preferably under
stringent conditions, to the upstream sequences of the open reading
frames shown in any one of SEQ ID NOs. 4, i.e. to the expression control
sequences shown in any one of SEQ ID NOs.: 1 to 3. Stringent
hybridization conditions as meant herein are, preferably, hybridization
conditions in 6× sodium chloride/sodium citrate (=SSC) at
approximately 45° C., followed by one or more wash steps in
0.2×SSC, 0.1% SDS at 53 to 65° C., preferably at 55°
C., 56° C., 57° C., 58° C., 59° C.,
60° C., 61° C., 62° C., 63° C., 64° C.
or 65° C. The skilled worker knows that these hybridization
conditions differ depending on the type of nucleic acid and, for example
when organic solvents are present, with regard to the temperature and
concentration of the buffer. For example, under "standard hybridization
conditions" the temperature differs depending on the type of nucleic acid
between 42° C. and 58° C. in aqueous buffer with a
concentration of 0.1 to 5×SSC (pH 7.2). If organic solvent is
present in the abovementioned buffer, for example 50% formamide, the
temperature under standard conditions is approximately 42° C. The
hybridization conditions for DNA:DNA hybrids are preferably for example
0.1×SSC and 20° C. to 45° C., preferably between
30° C. and 45° C. The hybridization conditions for DNA:RNA
hybrids are preferably, for example, 0.1×SSC and 30° C. to
55° C., preferably between 45° C. and 55° C. The
abovementioned hybridization temperatures are determined for example for
a nucleic acid with approximately 100 bp (=base pairs) in length and a
G+C content of 50% in the absence of formamide. Such hybridizing
expression control sequences are, more preferably, at least 70%, at least
80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%
at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identical to the expression control sequences as shown in any one of SEQ
ID NOs.: 1 to 3. The percent identity values are, preferably, calculated
over the entire nucleic acid sequence region. A series of programs based
on a variety of algorithms is available to the skilled worker for
comparing different sequences. In this context, the algorithms of
Needleman and Wunsch or Smith and Waterman give particularly reliable
results. To carry out the sequence alignments, the program PileUp (J.
Mol. Evolution., 25, 351-360, 1987, Higgins 1989, CABIOS, 5: 151-153) or
the programs Gap and BestFit (Needleman 1970 J. Mol. Biol. 48; 443-453
and Smith 1981, Adv. Appl. Math. 2; 482-489), which are part of the GCG
software packet (Genetics Computer Group, 575 Science Drive, Madison,
Wis., USA 53711 version 1991), are to be used. The sequence identity
values recited above in percent (%) are to be determined, preferably,
using the program GAP over the entire sequence region with the following
settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and
Average Mismatch: 0.000, which, unless otherwise specified, shall always
be used as standard settings for sequence alignments.

[0022] Moreover, expression control sequences which allow for
bidirectional expression can not only be found upstream of the
aforementioned open reading frames having a nucleic acid sequence as
shown in any one of SEQ ID NOs. 4. Rather, expression control sequences
which allow for seed specific expression can also be found upstream of
orthologous, paralogous or homologous genes (i.e. open reading frames).
Thus, also preferably, an variant expression control sequence comprised
by a polynucleotide of the present invention has a nucleic acid sequence
which hybridizes to a nucleic acid sequences located upstream of an open
reading frame sequence being at least 70%, more preferably, at least 80%,
at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identical to a sequence as shown in any one of SEQ ID NOs: 4. The said
variant open reading shall encode a polypeptide having the biological
activity of the corresponding polypeptide being encoded by the open
reading frame shown in any one of SEQ ID NOs.: 4. In this context it
should be mentioned that the open reading frame shown in SEQ ID NO: 4
encodes a polypeptide having the amino acid sequence shown in SEQ ID NO:
5 and, preferably, encodes a 60S acidic ribosomal protein P3.

[0023] Also preferably, a variant expression control sequence comprised by
a polynucleotide of the present invention is (i) obtainable by 5' genome
walking or TAIL PCR from an open reading frame sequence as shown in any
one of SEQ ID NOs: 4 or (ii) obtainable by 5' genome walking or TAIL PCR
from a open reading frame sequence being at least 80% identical to an
open reading frame as shown in any one of SEQ ID NOs: 4. Variant
expression control sequences are obtainable without further ado by the
genome walking technology or by thermal asymmetric interlaced polymerase
chain reaction (TAIL-PCR) which can be carried out as described in the
accompanying Examples by using, e.g., commercially available kits.

[0024] Variant expression control sequences referred to in this
specification for the expression control sequence shown in SEQ ID NOs: 1
to 3, preferably, comprise at least 10, at least 20, at least 30, at
least 40, at least 50 or all of the sequence motifs recited in Table 3.
More preferably, the variant expression control sequence comprises the
sequence motifs shown in any one of SEQ ID NOs: 54 to 76.

[0025] Also preferably, the expression control sequence comprised by the
polynucleotide of the present invention allows for a tissue specific
expression. Tissues in which the expression control sequence allows for
bidirectional specific expression are the following indicated tissues and
cells: 1) roots and leafs at 5-leaf stage, 2) stem at V-7 stage, 3)
Leaves, husk, and silk at flowering stage (at the first emergence of
silk), 4) Spikelets/Tassel at pollination, 5) Ear or Kernels at 5, 10,
15, 20, and 25 days after pollination.

[0026] More preferably, specific expression in the forward direction of
the expression control sequence of the present invention is in the seed,
preferably, whole seed, and the stem. Also more preferably, specific
expression in the reverse direction of the expression control sequence of
the present invention can be seen in leaf and root.

[0027] The term "specific" as used herein means that the nucleic acids of
interest being operatively linked to the expression control sequence
referred to herein will be predominantly expressed in the indicated
tissues or cells when present in a plant. A predominant expression as
meant herein is characterized by a statistically significantly higher
amount of detectable transcription in the said tissue or cells with
respect to other plant tissues. A statistically significant higher amount
of transcription is, preferably, an amount being at least two-fold,
three-fold, four-fold, five-fold, ten-fold, hundred-fold, five
hundred-fold or thousand-fold the amount found in at least one of the
other tissues with detectable transcription. Alternatively, it is an
expression in the indicated tissue or cell whereby the amount of
transcription in other tissues or cells is less than 1%, 2%, 3%, 4% or,
most preferably, 5% of the overall (whole plant) amount of expression.
The amount of transcription directly correlates to the amount of
transcripts (i.e. RNA) or polypeptides encoded by the transcripts present
in a cell or tissue. Suitable techniques for measuring transcription
either based on RNA or polypeptides are well known in the art. Tissue or
cell specificity alternatively and, preferably in addition to the above,
means that the expression is restricted or almost restricted to the
indicated tissue or cells, i.e. there is essentially no detectable
transcription in other tissues. Almost restricted as meant herein means
that unspecific expression is detectable in less than ten, less than
five, less than four, less than three, less than two or one other
tissue(s). Tissue or cell specific expression as used herein includes
expression in the indicated tissue or cells as well as in precursor
tissue or cells in the developing embryo.

[0028] An expression control sequences can be tested for tissue or cell
specific expression by determining the expression pattern of a nucleic
acid of interest, e.g., a nucleic acid encoding a reporter protein, such
as GFP, in a transgenic plant. Transgenic plants can be generated by
techniques well known to the person skilled in the art and as discussed
elsewhere in this specification. The aforementioned amounts or expression
pattern are, preferably, determined by Northern Blot or in situ
hybridization techniques as described in WO 02/102970. Preferred
expression pattern for the expression control sequences according to the
present invention are shown in the Figure or described in the
accompanying Examples, below.

[0029] The term "nucleic acid of interest" refers to a nucleic acid which
shall be expressed under the control of the expression control sequence
referred to herein. Preferably, a nucleic acid of interest encodes a
polypeptide the presence of which is desired in a cell or non-human
organism as referred to herein and, in particular, in a plant seed. Such
a polypeptide may be an enzyme which is required for the synthesis of
seed storage compounds or may be a seed storage protein. It is to be
understood that if the nucleic acid of interest encodes a polypeptide,
transcription of the nucleic acid in RNA and translation of the
transcribed RNA into the polypeptide may be required. A nucleic acid of
interest, also preferably, includes biologically active RNA molecules
and, more preferably, antisense RNAs, ribozymes, micro RNAs or siRNAs.
Said biologically active RNA molecules can be used to modify the amount
of a target polypeptide present in a cell or non-human organism. For
example, an undesired enzymatic activity in a seed can be reduced due to
the seed specific expression of an antisense RNAs, ribozymes, micro RNAs
or siRNAs. The underlying biological principles of action of the
aforementioned biologically active RNA molecules are well known in the
art. Moreover, the person skilled in the art is well aware of how to
obtain nucleic acids which encode such biologically active RNA molecules.
It is to be understood that the biologically active RNA molecules may be
directly obtained by transcription of the nucleic acid of interest, i.e.
without translation into a polypeptide. Preferably, at least one nucleic
acid of interest to be expressed under the control of the expression
control sequence of the present invention is heterologous in relation to
said expression control sequence, i.e. it is not naturally under the
control thereof, but said control has been produced in a non-natural
manner (for example by genetic engineering processes).

[0030] The term "operatively linked" as used herein means that the
expression control sequence of the present invention and a nucleic acid
of interest, are linked so that the expression can be governed by the
said expression control sequence, i.e. the expression control sequence
shall be functionally linked to the said nucleic acid sequence to be
expressed. Accordingly, the expression control sequence and, the nucleic
acid sequence to be expressed may be physically linked to each other,
e.g., by inserting the expression control sequence at the 5'end of the
nucleic acid sequence to be expressed. Alternatively, the expression
control sequence and the nucleic acid to be expressed may be merely in
physical proximity so that the expression control sequence is capable of
governing the expression of at least one nucleic acid sequence of
interest. The expression control sequence and the nucleic acid to be
expressed are, preferably, separated by not more than 500 bp, 300 bp, 100
bp, 80 bp, 60 bp, 40 bp, 20 bp, 10 by or 5 bp. For the bidirectional
expression control sequence of the present invention it is to e
understood that the above applies for both of the operatively the nucleic
acids of interest. It will be understood that non-essential sequences of
one of the expression control sequence of the invention can be deleted
without significantly impairing the properties mentioned. Delimitation of
the expression control sequence to particular essential regulatory
regions can also be undertaken with the aid of a computer program such as
the PLACE program ("Plant Cis-acting Regulatory DNA Elements") (Higo K et
al. (1999) Nucleic Acids Res 27:1, 297-300) or the BIOBASE database
"Transfac" (Biologische Datenbanken GmbH, Braunschweig). Processes for
mutagenizing nucleic acid sequences are known to the skilled worker and
include by way of example the use of oligonucleotides having one or more
mutations compared with the region to be mutated (e.g. within the
framework of a site-specific mutagenesis). Primers having approximately
15 to approximately 75 nucleotides or more are typically employed, with
preferably about 10 to about 25 or more nucleotide residues being located
on both sides of the sequence to be modified. Details and procedure for
said mutagenesis processes are familiar to the skilled worker (Kunkel et
al. (1987) Methods Enzymol 154:367-382; Tomic et al. (1990) Nucl Acids
Res 12:1656; Upender et al. (1995) Biotechniques 18(1):29-30; U.S. Pat.
No. 4,237,224). A mutagenesis can also be achieved by treatment of, for
example, vectors comprising one of the nucleic acid sequences of the
invention with mutagenizing agents such as hydroxylamine.

[0031] Advantageously, it has been found in the studies underlying the
present invention that bidirectional expression of two nucleic acids of
interest can be achieved by expressing said nucleic acids of interest
under the control of an expression control sequence from maize or a
variant expression control sequence as specified above. The expression
control sequences provided by the present invention allow for a reliable
bidirectional expression of nucleic acids of interest. Thanks to the
present invention, it is possible to (i) specifically manipulate
biochemical processes in specific tissues, e.g., by expressing
heterologous enzymes or biologically active RNAs, or (ii) to produce
heterologous proteins in said tissues, or (iii) to provide nucleic acids
of interest in a stoichiometric ratio. In principle, the present
invention contemplates the use of the polynucleotide, the vector, the
host cell or the non-human transgenic organism for the expression of a
nucleic acid of interest. The invention makes it possible to increase the
number of transcription units with a reduced number of promoter
sequences. In the case of translation fusions it is also possible to
regulate more than two proteins. A particular advantage of this invention
is that the expression of these multiple transgenes takes place
simultaneously and synchronously under the control of the bidirectional
promoter. The promoter is particularly suitable for coordinating
expression of nucleic acids. Thus, it is possible to express
simultaneously: (i) target protein and selection marker or reporter
protein, ii) selection marker and reporter protein, iii) two target
proteins, e.g. from the same metabolic pathway iii) sense and antisense
RNA, iv) various proteins for defense against pathogens, and many more,
and v) bring about improved effects in the plants.

[0032] The present invention also relates to a vector comprising the
polynucleotide of the present invention.

[0033] The term "vector", preferably, encompasses phage, plasmid, viral or
retroviral vectors as well as artificial chromosomes, such as bacterial
or yeast artificial chromosomes. Moreover, the term also relates to
targeting constructs which allow for random or site-directed integration
of the targeting construct into genomic DNA. Such target constructs,
preferably, comprise DNA of sufficient length for either homologous or
heterologous recombination as described in detail below. The vector
encompassing the polynucleotides of the present invention, preferably,
further comprises selectable markers for propagation and/or selection in
a host. The vector may be incorporated into a host cell by various
techniques well known in the art. If introduced into a host cell, the
vector may reside in the cytoplasm or may be incorporated into the
genome. In the latter case, it is to be understood that the vector may
further comprise nucleic acid sequences which allow for homologous
recombination or heterologous insertion. Vectors can be introduced into
prokaryotic or eukaryotic cells via conventional transformation or
transfection techniques. The terms "transformation" and "transfection",
conjugation and transduction, as used in the present context, are
intended to comprise a multiplicity of prior-art processes for
introducing foreign nucleic acid (for example DNA) into a host cell,
including calcium phosphate, rubidium chloride or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofection,
natural competence, carbon-based clusters, chemically mediated transfer,
electroporation or particle bombardment (e.g., "gene-gun"). Suitable
methods for the transformation or transfection of host cells, including
plant cells, can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other
laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44,
Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa,
N.J. Alternatively, a plasmid vector may be introduced by heat shock or
electroporation techniques. Should the vector be a virus, it may be
packaged in vitro using an appropriate packaging cell line prior to
application to host cells. Retroviral vectors may be replication
competent or replication defective. In the latter case, viral propagation
generally will occur only in complementing host/cells.

[0034] Preferably, the vector referred to herein is suitable as a cloning
vector, i.e. replicable in microbial systems. Such vectors ensure
efficient cloning in bacteria and, preferably, yeasts or fungi and make
possible the stable transformation of plants. Those which must be
mentioned are, in particular, various binary and co-integrated vector
systems which are suitable for the T-DNA-mediated transformation. Such
vector systems are, as a rule, characterized in that they contain at
least the vir genes, which are required for the Agrobacterium-mediated
transformation, and the sequences which delimit the T-DNA (T-DNA border).
These vector systems, preferably, also comprise further cis-regulatory
regions such as promoters and terminators and/or selection markers with
which suitable transformed host cells or organisms can be identified.
While co-integrated vector systems have vir genes and T-DNA sequences
arranged on the same vector, binary systems are based on at least two
vectors, one of which bears vir genes, but no T-DNA, while a second one
bears T-DNA, but no vir gene. As a consequence, the last-mentioned
vectors are relatively small, easy to manipulate and can be replicated
both in E. coli and in Agrobacterium. These binary vectors include
vectors from the pBIB-HYG, pPZP, pBecks, pGreen series. Preferably used
in accordance with the invention are Bin19, pBI101, pBinAR, pGPTV, pSUN
and pCAMBIA. An overview of binary vectors and their use can be found in
Hellens et al, Trends in Plant Science (2000) 5, 446-451. Furthermore, by
using appropriate cloning vectors, the polynucleotide of the invention
can be introduced into host cells or organisms such as plants or animals
and, thus, be used in the transformation of plants, such as those which
are published, and cited, in: Plant Molecular Biology and Biotechnology
(CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F.
White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants,
vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press,
1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in:
Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R.
Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol.
Plant Molec. Biol. 42 (1991), 205-225.

[0035] More preferably, the vector of the present invention is an
expression vector. In such an expression vector, the polynucleotide
comprises an expression cassette as specified above allowing for
expression in eukaryotic cells or isolated fractions thereof. An
expression vector may, in addition to the polynucleotide of the
invention, also comprise further regulatory elements including
transcriptional as well as translational enhancers. Preferably, the
expression vector is also a gene transfer or targeting vector. Expression
vectors derived from viruses such as retroviruses, vaccinia virus,
adeno-associated virus, herpes viruses, or bovine papilloma virus, may be
used for delivery of the polynucleotides or vector of the invention into
targeted cell population. Methods which are well known to those skilled
in the art can be used to construct recombinant viral vectors; see, for
example, the techniques described in Sambrook, Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel,
Current Protocols in Molecular Biology, Green Publishing Associates and
Wiley Interscience, N.Y. (1994).

[0036] Suitable expression vector backbones are, preferably, derived from
expression vectors known in the art such as Okayama-Berg cDNA expression
vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogene) or
pSPORT1 (GIBCO BRL). Further examples of typical fusion expression
vectors are pGEX (Pharmacia Biotech Inc; Smith, D. B., and Johnson, K. S.
(1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and
pRIT5 (Pharmacia, Piscataway, N.J.), where glutathione S-transferase
(GST), maltose E-binding protein and protein A, respectively, are fused
with the nucleic acid of interest encoding a protein to be expressed. The
target gene expression of the pTrc vector is based on the transcription
from a hybrid trp-lac fusion promoter by host RNA polymerase. The target
gene expression from the pET 11d vector is based on the transcription of
a T7-gn10-lac fusion promoter, which is mediated by a coexpressed viral
RNA polymerase (T7 gn1). This viral polymerase is provided by the host
strains BL21 (DE3) or HMS174 (DE3) from a resident λ-prophage which
harbors a T7 gn1 gene under the transcriptional control of the lacUV 5
promoter. Examples of vectors for expression in the yeast S. cerevisiae
comprise pYepSecl (Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjan
and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987)
Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.).
Vectors and processes for the construction of vectors which are suitable
for use in other fungi, such as the filamentous fungi, comprise those
which are described in detail in: van den Hondel, C. A. M. J. J., & Punt,
P. J. (1991) "Gene transfer systems and vector development for
filamentous fungi, in: Applied Molecular Genetics of fungi, J. F. Peberdy
et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More
Gene Manipulations in Fungi (J. W. Bennett & L. L. Lasure, Ed., pp.
396-428: Academic Press: San Diego). Further suitable yeast vectors are,
for example, pAG-1, YEp6, YEp13 or pEMBLYe23. As an alternative, the
polynucleotides of the present invention can be also expressed in insect
cells using baculovirus expression vectors. Baculovirus vectors which are
available for the expression of proteins in cultured insect cells (for
example Sf9 cells) comprise the pAc series (Smith et al. (1983) Mol. Cell
Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology 170:31-39).

[0037] The polynucleotides of the present invention can be used for
expression of a nucleic acid of interest in single-cell plant cells (such
as algae), see Falciatore et al., 1999, Marine Biotechnology 1
(3):239-251 and the references cited therein, and plant cells from higher
plants (for example Spermatophytes, such as arable crops) by using plant
expression vectors. Examples of plant expression vectors comprise those
which are described in detail in: Becker, D., Kemper, E., Schell, J., and
Masterson, R. (1992) "New plant binary vectors with selectable markers
located proximal to the left border", Plant Mol. Biol. 20:1195-1197; and
Bevan, M. W. (1984) "Binary Agrobacterium vectors for plant
transformation", Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer
in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and
Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, p. 15-38. A plant
expression cassette, preferably, comprises regulatory sequences which are
capable of controlling the gene expression in plant cells and which are
functionally linked so that each sequence can fulfill its function, such
as transcriptional termination, for example polyadenylation signals.
Preferred polyadenylation signals are those which are derived from
Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid
pTiACH5, which is known as octopine synthase (Gielen et al., EMBO J. 3
(1984) 835 et seq.) or functional equivalents of these, but all other
terminators which are functionally active in plants are also suitable.

[0038] Since plant gene expression is very often not limited to
transcriptional levels, a plant expression cassette preferably comprises
other functionally linked sequences such as translation enhancers, for
example the overdrive sequence, which comprises the 5'-untranslated
tobacco mosaic virus leader sequence, which increases the protein/RNA
ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711). Other
preferred sequences for the use in functional linkage in plant gene
expression cassettes are targeting sequences which are required for
targeting the gene product into its relevant cell compartment (for a
review, see Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and
references cited therein), for example into the vacuole, the nucleus, all
types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the
extracellular space, the mitochondria, the endoplasmic reticulum, oil
bodies, peroxisomes and other compartments of plant cells.

[0039] The abovementioned vectors are only a small overview of vectors to
be used in accordance with the present invention. Further vectors are
known to the skilled worker and are described, for example, in: Cloning
Vectors (Ed., Pouwels, P. H., et al., Elsevier, Amsterdam-New
York-Oxford, 1985, ISBN 0 444 904018). For further suitable expression
systems for prokaryotic and eukaryotic cells see the chapters 16 and 17
of Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0040] The present invention also contemplates a host cell comprising the
polynucleotide or the vector of the present invention.

[0041] Host cells are primary cells or cell lines derived from
multicellular organisms such as plants or animals. Furthermore, host
cells encompass prokaryotic or eukaryotic single cell organisms (also
referred to as micro-organisms). Primary cells or cell lines to be used
as host cells in accordance with the present invention may be derived
from the multicellular organisms referred to below. Host cells which can
be exploited are furthermore mentioned in: Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Specific expression strains which can be used, for example those
with a lower protease activity, are described in: Gottesman, S., Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San
Diego, Calif. (1990) 119-128. These include plant cells and certain
tissues, organs and parts of plants in all their phenotypic forms such as
anthers, fibers, root hairs, stalks, embryos, calli, cotelydons,
petioles, harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or can be
used for bringing about the transgenic plant. Preferably, the host cells
may be obtained from plants. More preferably, oil crops are envisaged
which comprise large amounts of lipid compounds, such as oilseed rape,
evening primrose, hemp, thistle, peanut, canola, linseed, soybean,
safflower, sunflower, borage, or plants such as maize, wheat, rye, oats,
triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae
plants such as potato, tobacco, eggplant and tomato, Vicia species, pea,
alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil
palm, coconut) and perennial grasses and fodder crops. Especially
preferred plants according to the invention are oil crops such as
soybean, peanut, oilseed rape, canola, linseed, hemp, evening primrose,
sunflower, safflower, trees (oil palm, coconut). Suitable methods for
obtaining host cells from the multicellular organisms referred to below
as well as conditions for culturing these cells are well known in the
art.

[0043] How to culture the aforementioned micro-organisms is well known to
the person skilled in the art.

[0044] The present invention also relates to a non-human transgenic
organism, preferably a plant or seed thereof, comprising the
polynucleotide or the vector of the present invention.

[0045] The term "non-human transgenic organism", preferably, relates to a
plant, a plant seed, a non-human animal or a multicellular
micro-organism. The polynucleotide or vector may be present in the
cytoplasm of the organism or may be incorporated into the genome either
heterologous or by homologous recombination. Host cells, in particular
those obtained from plants or animals, may be introduced into a
developing embryo in order to obtain mosaic or chimeric organisms, i.e.
non-human transgenic organisms comprising the host cells of the present
invention. Suitable transgenic organisms are, preferably, all organisms
which are suitable for the expression of recombinant genes.

[0049] Preferably, a multicellular micro-organism as used herein refers to
protists or diatoms. More preferably, it is selected from the group of
the families Dinophyceae, Turaniellidae or Oxytrichidae, such as the
genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum,
Stylonychia mytilus, Stylonychia pustulate, Stylonychia putrina,
Stylonychia notophora, Stylonychia sp., Colpidium campylum or Colpidium
sp.

[0050] The present invention also relates to a method for expressing a
nucleic acid of interest in a host cell comprising [0051] (a)
introducing the polynucleotide or the vector of the present invention
into the host cell; and [0052] (b) expressing at least one nucleic acid
of interest in said host cell.

[0053] The polynucleotide or vector of the present invention can be
introduced into the host cell by suitable transfection or transformation
techniques as specified elsewhere in this description. The nucleic acid
of interest will be expressed in the host cell under suitable conditions.
To this end, the host cell will be cultivated under conditions which, in
principle, allow for transcription of nucleic acids. Moreover, the host
cell, preferably, comprises the exogenously supplied or endogenously
present transcription machinery required for expressing a nucleic acid of
interest by the expression control sequence. More preferably, expressing
in the method of the present invention refers to bidirectional expression
of at least one nucleic acid of interest in each of the two orientations
from the expression control sequence.

[0054] Moreover, the present invention encompasses a method for expressing
a nucleic acid of interest in a non-human organism comprising [0055]
(a) introducing the polynucleotide or the vector of the present invention
into the non human organism; and [0056] (b) expressing at least one
nucleic acid of interest in said non-human transgenic organism.

[0057] The polynucleotide or vector of the present invention can be
introduced into the non-human transgenic organism by suitable techniques
as specified elsewhere in this description. The non-human transgenic
organism, preferably, comprises the exogenously supplied or endogenously
present transcription machinery required for expressing a nucleic acid of
interest by the expression control sequence. More preferably, expressing
in the method of the present invention refers to bidirectional expression
of at least one nucleic acid of interest in each of the two orientations
from the expression control sequence.

[0058] In the following, some preferred embodiments pertaining to the
present invention are described in more detail.

[0059] In a preferred embodiment, the polynucleotide of the present
invention also comprises further genetic control sequences. A genetic
control sequence as referred to in accordance with the present invention
is to be understood broadly and means all sequences having an influence
on the coming into existence of the function of the transgenic expression
cassette of the invention. Genetic control sequences modify for example
the transcription and translation in prokaryotic or eukaryotic organisms.
The expression cassettes of the invention preferably comprise as
additional genetic control sequence one of the promoters of the invention
5'-upstream from the particular nucleic acid sequence to be expressed
transgenically, and a terminator sequence 3'-downstream, and if
appropriate further usual regulatory elements, in each case functionally
linked to the nucleic acid sequence to be expressed transgenically.

[0060] Genetic control sequences also comprise further promoters, promoter
elements or minimal promoters which are able to modify the
expression-controlling properties. It is thus possible for example
through genetic control sequences for tissue-specific expression to take
place additionally in dependence on particular stress factors.
Corresponding elements are described for example for water stress,
abscisic acid (Lam E and Chua N H, (1991) J Biol Chem
266(26):17131-17135) and heat stress (Schoffl F et al. (1989) Mol Gen
Genetics 217(2-3):246-53). A further possibility is for further promoters
which make expression possible in further plant tissues or in other
organisms such as, for example, E. coli bacteria to be functionally
linked to the nucleic acid sequence to be expressed. Suitable plant
promoters are in principle all the promoters described above. It is
conceivable for example that a particular nucleic acid sequence is
described by a promoter (for example one of the promoters of the
invention) in one plant tissue as sense RNA and translated into the
corresponding protein, while the same nucleic acid sequence is
transcribed by another promoter with a different specificity in a
different tissue into antisense RNA, and the corresponding protein is
down-regulated. This can be implemented by an expression cassette of the
invention by the one promoter being positioned in front of the nucleic
acid sequence to be expressed transgenically, and the other promoter
behind.

[0061] Genetic control sequences further comprise also the 5'-untranslated
region, introns or the noncoding 3' region of genes, preferably of the
pFD gene and/or of the OASTL gene. It has been shown that untranslated
regions may play a significant functions in the regulation of gene
expression. Thus, it has been shown that 5'-untranslated sequences may
enhance the transient expression of heterologous genes. They may moreover
promote tissue specificity (Rouster J et al. (1998) Plant J.
15:435-440.). Conversely, the 5'-untranslated region of the opaque-2 gene
suppresses expression. Deletion of the corresponding region leads to an
increase in gene activity (Lohmer S et al. (1993) Plant Cell 5:65-73).
Further 5'-untranslated sequences and introns with expression-promoting
function are known to the skilled worker. McElroy and coworkers (McElroy
et al. (1991) Mol Gen Genet 231(1):150-160) reported on a construct based
on the rice actin 1 (Act1) promoter for transforming monocotyledonous
plants. Use of the Act1 intron in combination with the 35S promoter in
transgenic rice cells led to an expression rate which was increased
ten-fold compared with the isolated 35S promoter. Optimization of the
sequence environment of the translation initiation site of the reporter
gene gene (GUS) resulted in a four-fold increase in GUS expression in
transformed rice cells. Combination of the optimized translation
initiation site and of the Act1 intron resulted in a 40-fold increase in
GUS expression by the CaMV35S promoter in transformed rice cells; similar
results have been obtained with transformed corn cells. Overall, it was
concluded from the investigations described above that the expression
vectors based on the Act1 promoter are suitable for controlling
sufficiently strong and constitutive expression of foreign DNA in
transformed cells of monocotyledonous plants.

[0062] The expression cassette may comprise one or more so-called enhancer
sequences functionally linked to the promoter, which make increased
transgenic expression of the nucleic acid sequence possible. It is also
possible to insert additional advantageous sequences, such as further
regulatory elements or terminators, at the 3' end of the nucleic acid
sequences which are to be expressed transgenically.

[0063] Control sequences additionally mean those which make homologous
recombination or insertion into the genome of a host organism possible or
which allow deletion from the genome. It is possible in homologous
recombination for example for the natural promoter of a particular gene
to be replaced by one of the promoters of the invention. Methods such as
the creaox technology permit tissue-specific deletion, which is inducible
in some circumstances, of the expression cassette from the genome of the
host organism (Sauer B. (1998) Methods. 14(4):381-92). In this case,
particular flanking sequences are attached (lox sequences) to the target
gene and subsequently make deletion possible by means of cre recombinase.
The promoter to be introduced can be placed by means of homologous
recombination in front of the target gene which is to be expressed
transgenically by linking the promoter to DNA sequences which are, for
example, homologous to endogenous sequences which precede the reading
frame of the target gene. Such sequences are to be regarded as genetic
control sequences. After a cell has been transformed with the appropriate
DNA construct, the two homologous sequences can interact and thus place
the promoter sequence at the desired site in front of the target gene, so
that the promoter sequence is now functionally linked to the target gene
and forms an expression cassette of the invention. The selection of the
homologous sequences determines the promoter insertion site. It is
possible in this case for the expression cassette to be generated by
homologous recombination by means of single or double reciprocal
recombination. In single reciprocal recombination there is use of only a
single recombination sequence, and the complete introduced DNA is
inserted. In double reciprocal recombination the DNA to be introduced is
flanked by two homologous sequences, and the flanking region is inserted.
The latter process is suitable for replacing, as described above, the
natural promoter of a particular gene by one of the promoters of the
invention and thus modifying the location and timing of gene expression.
This functional linkage represents an expression cassette of the
invention. To select successfully homologously recombined or else
transformed cells it is usually necessary additionally to introduce a
selectable marker. Various suitable markers are mentioned below. The
selection marker permits selection of transformed from untransformed
cells. Homologous recombination is a relatively rare event in higher
eukaryotes, especially in plants. Random integrations into the host
genome predominate. One possibility of deleting randomly integrated
sequences and thus enriching cell clones having a correct homologous
recombination consists of using a sequence-specific recombination system
as described in U.S. Pat. No. 6,110,736.

[0065] In a particularly preferred embodiment, the expression cassette
comprises a terminator sequence which is functional in plants. Terminator
sequences which are functional in plants means in general sequences able
to bring about termination of transcription of a DNA sequence in plants.
Examples of suitable terminator sequences are the OCS (octopine synthase)
terminator and the NOS (nopaline synthase) terminator. However, plant
terminator sequences are particularly preferred. Plant terminator
sequences means in general sequences which are a constituent of a natural
plant gene. Particular preference is given in this connection to the
terminator of the potato cathepsin D inhibitor gene (GenBank Acc. No.:
X74985) or of the terminator of the field bean storage protein gene
VfLEIB3 (GenBank Acc. No.: Z26489). These terminators are at least
equivalent to the viral or T-DNA terminators described in the art.

[0066] The skilled worker is also aware of a large number of nucleic acids
and proteins whose recombinant expression is advantageous under the
control of the expression cassettes or processes of the invention. The
skilled worker is further aware of a large number of genes through whose
repression or switching off by means of expression of an appropriate
antisense RNA it is possible likewise to achieve advantageous effects.
Non-restrictive examples of advantageous effects which may be mentioned
are: facilitated production of a transgenic organism for example through
the expression of selection markers, achievement of resistance to abiotic
stress factors (heat, cold, aridity, increased moisture, environmental
toxins, UV radiation), achievement of resistance to biotic stress factors
(pathogens, viruses, insects and diseases), improvement in human or
animal food properties, improvement in the growth rate of the yield. Some
specific examples of nucleic acids whose expression provides the desired
advantageous effects may be mentioned below:

[0067] 1. Selection Markers. Selection marker comprises both positive
selection markers which confer resistance to an antibiotic, herbicide or
biocide, and negative selection markers which confer sensitivity to
precisely the latter, and markers which provide the transformed organism
with a growth advantage (for example through expression of key genes of
cytokine biosynthesis; Ebinuma H et al. (2000) Proc Natl Acad Sci USA
94:2117-2121). In the case of positive selection, only the organisms
which express the corresponding selection marker thrive, whereas in the
case of negative selection it is precisely these which perish. The use of
a positive selection marker is preferred in the production of transgenic
plants.

[0069] The concentrations used in each case for the selection of
antibiotics, herbicides, biocides or toxins must be adapted to the
particular test conditions or organisms. Examples which may be mentioned
for plants are kanamycin (Km) 50 mgA, hygromycin B 40 mg/l,
phosphinothricin (ppt) 6 mgA. It is also possible to express functional
analogs of said nucleic acids coding for selection markers. Functional
analogs means in this connection all the sequences which have
substantially the same function, i.e. are capable of selecting
transformed organisms. It is moreover perfectly possible for the
functional analog to differ in other features. It may for example have a
higher or lower activity or else possess further functionalities.

[0071] 3. Expression of metabolic enzymes for use in the animal and human
food sectors, for example expression of phytase and cellulases.
Particular preference is given to nucleic acids such as the artificial
cDNA coding for a microbial phytase (GenBank Acc. No.: A19451) or
functional equivalents thereof.

[0072] 4. Achievement of resistance for example to fungi, insects,
nematodes and diseases through targeted secretion or accumulation of
particular metabolites or proteins in the epidermis of the embryo.
Examples which may be mentioned are glucosinolates (defense against
herbivors), chitinases or glucanases and other enzymes which destroy the
cell wall of parasites, ribosome-inactivating proteins (RIPs) and other
proteins of the plants' resistance and stress responses, as are induced
on injury or microbial attack of plants or chemically by, for example,
salicylic acid, jasmonic acid or ethylene, lysozymes from non-plant
sources such as, for example, T4 lysozyme or lysozyme from various
mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin,
[alpha]-amylase inhibitor or protease inhibitors (cowpea trypsin
inhibitor), glucanases, lectins such as phytohemagglutinin, wheatgerm
agglutinin, RNAses or ribozymes. Particularly preferred nucleic acids are
those coding for the chit42 endochitinase from Trichoderma harzianum
(GenBank Acc. No.: S78423) or for the N-hydroxylating, multi-functional
cytochrome P-450 (CYP79) proteins from Sorghum bicolor (GenBank Acc. No.:
U32624) or functional equivalents thereof.

[0073] 5. The accumulation of glucosinolates in plants of the Cardales
genus, especially the oil seeds to protect from pests (Rask L et al.
(2000) Plant Mol Biol 42:93-113; Menard R et al. (1999) Phytochemistry
52:29-35), expression of the Bacillus thuringiensis endotoxin under the
control of the 35S CaMV promoter (Vaeck et al. (1987) Nature 328:33-37)
or protection of tobacco against fungal attack by expression of a bean
chitonase under the control of the CaMV promoter (Broglie et al. (1991)
Science 254:1194-119, is known.

[0075] 6. Expression of genes which bring about accumulation of fine
chemicals such as of tocopherols, tocotrienols or carotenoids. An example
which may be mentioned is phytoene desaturase. Nucleic acids which code
for the phytoene desaturase from Narcissus pseudonarcissus (GenBank Acc.
No.: X78815) or functional equivalents thereof are preferred.

[0078] 9. Achieving an increased storage ability in cells which normally
comprise few storage proteins or lipids with the aim of increasing the
yield of these substances, for example by expression of an acetyl-CoA
carboxylase. Preferred nucleic acids are those which code for the
acetyl-CoA carboxylase (accase) from Medicago sativa (GenBank Acc. No.:
L25042) or functional equivalents thereof. Further examples of
advantageous genes are mentioned for example in Dunwell J M (2000) J Exp
Bot. 51 Spec No:487-96.

[0079] It is also possible to express functional analogs of said nucleic
acids and proteins. Functional analogs means in this connection all the
sequences which have substantially the same function, i.e. are capable of
the function (for example a substrate conversion or signal transduction)
like the protein mentioned by way of example too. It is moreover
perfectly possible for the functional analog to differ in other features.
It may for example have a higher or lower activity or else possess
further functionalities. Functional analogs also means sequences which
code for fusion proteins consisting of one of the preferred proteins and
other proteins, for example a further preferred protein or else a signal
peptide sequence.

[0080] Expression of the nucleic acids under the control of the promoters
of the invention is possible in any desired cell compartment such as, for
example, the endomembrane system, the vacuole and the chloroplasts.
Desired glycosylation reactions, especially foldings and the like, are
possible by utilizing the secretory pathway. Secretion of the target
protein to the cell surface or secretion into the culture medium, for
example on use of suspension-cultured cells or protoplasts, is also
possible. The target sequences necessary for this purpose can thus be
taken into account in individual vector variations and be introduced,
together with the target gene to be cloned, into the vector through use
of a suitable cloning strategy. It is possible to utilize as target
sequences both gene-intrinsic, where present, or heterologous sequences.
Additional heterologous sequences which are preferred for the functional
linkage, but not restricted thereto, are further targeting sequences to
ensure the subcellular localization in apoplasts, in the vacuole, in
plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the
cell nucleus, in elaioplasts or other compartments; and translation
enhancers' such as the 5' leader sequence from tobacco mosaic virus
(Gallie et al. (1987) Nucl Acids Res 15 8693-8711) and the like. The
process for transporting proteins which are not localized per se in the
plastids in a targeted fashion into the plastids is described (Klosgen R
B & Weil J H (1991) Mol Gen Genet 225(2):297-304; Van Breusegem F et al.
(1998) Plant Mol Biol 38(3):491-496). Preferred sequences are

[0085] The target sequences may be linked to other target sequences which
differ from the transit peptide-encoding sequences in order to ensure a
subcellular localization in the apoplast, in the vacuole, in plastids, in
the mitochondrion, in the endoplasmic reticulum (ER), in the cell
nucleus, in elaioplasts or other compartments. It is also possible to
employ translation enhancers such as the 5' leader sequence from tobacco
mosaic virus (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the
like.

[0086] The skilled worker is also aware that he need not express the genes
described above directly by use of the nucleic acid sequences coding for
these genes, or repress them for example by anti-sense. He can also use
for example artificial transcription factors of the type of zinc finger
proteins (Beerli R R et al. (2000) Proc Natl Acad Sci USA
97(4):1495-500). These factors bind in the regulatory regions of the
endogenous genes which are to be expressed or repressed and result,
depending on the design of the factor, in expression or repression of the
endogenous gene. Thus, the desired effects can also be achieved by
expression of an appropriate zinc finger transcription factor under the
control of one of the promoters of the invention.

[0087] The expression cassettes of the invention can likewise be employed
for suppressing or reducing replication or/and translation of target
genes by gene silencing.

[0088] The expression cassettes of the invention can also be employed for
expressing nucleic acids which mediate so-called antisense effects and
are thus able for example to reduce the expression of a target protein.

[0089] Preferred genes and proteins whose suppression is the condition for
an advantageous phenotype comprise by way of example, but
non-restrictively:

[0090] a) polygalacturonase to prevent cell degradation and mushiness of
plants and fruits, tomatoes for example. Preferably used for this purpose
are nucleic acid sequences such as that of the tomato polygalacturonase
gene (Gen Bank Acc. No.: X14074) or its homologs from other genera and
species.

[0093] d) shifting the amylose/amylopectin content in starch by
suppression of branching enzyme Q, which is responsible for
[alpha]-1,6-glycosidic linkage. Corresponding procedures are described
(for example in Schwall G P et al. (2000) Nat Biotechnol 18(5):551-554).
Preferably used for this purpose are nucleic acid sequences like that of
the starch branching enzyme II of potato (GenBank Acc. No.: AR123356;
U.S. Pat. No. 6,169,226) or its homologs from other genera and species.

[0094] An "antisense" nucleic acid means primarily a nucleic acid sequence
which is wholly or partly complementary to at least part of the sense
strand of said target protein. The skilled worker is aware that he can
use alternatively the cDNA or the corresponding gene as starting template
for corresponding antisense constructs. The antisense nucleic acid is
preferably complementary to the coding region of the target protein or a
part thereof. The antisense nucleic acid may, however, also be
complementary to the non-coding region of a part thereof. Starting from
the sequence information for a target protein, an antisense nucleic acid
can be designed in a manner familiar to the skilled worker by taking
account of the base-pair rules of Watson and Crick. An antisense nucleic
acid may be complementary to the whole or a part of the nucleic acid
sequence of a target protein. In a preferred embodiment, the antisense
nucleic acid is an oligonucleotide with a length of for example 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides.

[0095] The antisense nucleic acid comprises in a preferred embodiment
[alpha]-anomeric nucleic acid molecules. [alpha]-Anomeric nucleic acid
molecules form in particular double-stranded hybrids with complementary
RNA in which the strands run parallel to one another, in contrast to the
normal [beta] units (Gaultier et al. (1987) Nucleic Acids Res
15:6625-6641). The use of the sequences described above in sense
orientation is likewise encompassed and may, as is familiar to the
skilled worker, lead to cosuppression. The expression of sense RNA to an
endogenous gene may reduce or switch off its expression, similar to that
described for antisense approaches (Goring et al. (1991) Proc Natl Acad
Sci USA 88:1770-1774; Smith et al. (1990) Mol Gen Genet 224:447-481;
Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol et al. (1990)
Plant Cell 2:291-299). It is moreover for the introduced construct to
represent the gene to be reduced wholly or only in part. The possibility
of translation is unnecessary.

[0096] It is also very particularly preferred to use processes such as
gene regulation by means of double-stranded RNA (double-stranded RNA
interference). Corresponding processes are known to the skilled worker
and described in detail (e.g. Matzke M A et al. (2000) Plant Mol Biol
43:401-415; Fire A. et al (1998) Nature 391:806-811; WO 99/32619; WO
99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO
00/63364). Express reference is made to the processes and methods
described in the indicated references. Highly efficient suppression of
native genes is brought about here through simultaneous introduction of
strand and complementary strand.

[0099] The bidirectional promoters of the invention are particularly
advantageous when it is employed for regulating two enzymes of a
metabolic pathway. 2'-Methyl-6-phytylhydroquinone methyltransferase and
homogentisate phytyl-pyrophosphate-transferase, for example, can be
expressed simultaneously via one of the bidirectional promoters of the
invention, bringing about an increase in tocopherols. In addition,
inhibition of homogentisate dioxygenase (for example by expression of a
corresponding dsRNA) and overexpression of tyrosine aminotransferase
leads to an increase in the tocopherol content. In carotenoid metabolism,
inhibition of [alpha]-cyclase and overexpression of [beta]-cyclase leads
to a change in the content of [alpha]-carotene and [beta]-carotene.

[0100] It is possible to prevent post-transcriptional silencing effects by
parallel inhibition of the transcription of the SDE3 gene and
overexpression of the recombinant protein (WO 02/063039).

[0101] Immunologically active parts of antibodies can also be
advantageously expressed by using the promoters of the invention. Thus,
for example, the heavy chain of an IgG1 antibody can be expressed in one
direction, and the light chain in the other direction. The two form a
functional antibody after translation (WO 02/101006).

[0102] A further possibility is to express simultaneously stress-related
ion transporters (WO 03/057899) together with herbicide genes in order to
increase the tolerance of environmental effects.

[0103] Many enzymes consist of two or more subunits, both of which are
necessary for functioning. It is possible by means of one of the
bidirectional promoters of the invention to express two subunits
simultaneously. One example thereof is overexpression of the [alpha] and
[beta] subunits of follicle stimulating human hormone.

[0104] A construct consisting of a gene for a selection marker and a
reporter gene is particularly valuable for establishing transformation
systems, when they are regulated by this bidirectional promoter.

[0105] The expression cassettes of the invention and the vectors derived
therefrom may comprise further functional elements. The term functional
element is to be understood broadly and means all elements which have an
influence on production, multiplication or function of the expression
cassettes of the invention or vectors or organisms derived therefrom.
Non-restrictive examples which may be mentioned are:

[0108] c) Elements for example "border sequences" which make
agrobacteria-mediated transfer into plant cells possible for transfer and
integration into the plant genome, such as, for example, the right or
left border of the T-DNA or the vir region.

[0109] d) Multiple cloning regions (MCS) permit and facilitate the
insertion of one or more nucleic acid sequences.

[0110] The skilled worker is aware of various ways of obtaining an
expression cassette of the invention. The production of an expression
cassette of the invention takes place for example by fusing one of the
expression control sequence of the invention with a nucleic acid sequence
of interest to be expressed, if appropriate with a sequence coding for a
transit peptide, preferably a chloroplast-specific transit peptide which
is preferably disposed between the promoter and the respective nucleic
acid sequence, and with a terminator or polyadenylation signal.
Conventional techniques of recombination and cloning are used for this
purpose (as described above).

[0111] However, and expression cassette also means constructions in which
the promoter, without previously having been functionally linked to a
nucleic acid sequence to be expressed, is introduced into a host genome,
for example via a targeted homologous recombination or a random
insertion, there assumes regulatory control of nucleic acid sequences
which are then functionally linked to it, and controls transgenic
expression thereof. Insertion of the promoter-for example by homologous
recombination-in front of a nucleic acid coding for a particular
polypeptide results in an expression cassette of the invention which
controls the expression of the particular polypeptide in the plant. The
insertion of the promoter may also take place by expression of antisense
RNA to the nucleic acid coding for a particular polypeptide. Expression
of the particular polypeptide in plants is thus downregulated or switched
off.

[0112] It is also possible analogously for a nucleic acid sequence to be
expressed transgenically to be placed, for example by homologous
recombination, behind the endogenous, natural promoter, resulting in an
expression cassette of the invention which controls the expression of the
nucleic acid sequence to be expressed transgenically.

[0113] In principle, the invention also contemplates cells, cell cultures,
parts-such as, for example, roots, leaves etc. in the case of transgenic
plant organisms and transgenic propagation material such as seeds or
fruits, derived from the transgenic organisms described above.

[0114] Genetically modified plants of the invention which can be consumed
by humans and animals may also be used as human food or animal food for
example directly or after processing in a manner known per se.

[0115] A further aspect of the invention, thus, relates to the use of the
transgenic organisms of the invention described above and of the cells,
cell cultures, parts-such as, for example, roots, leaves etc. in the case
of transgenic plant organisms-and transgenic propagation material such as
seeds or fruits derived therefrom for producing human or animal foods,
pharmaceuticals or fine chemicals.

[0116] Preference is further given to a process for the recombinant
production of pharmaceuticals or fine chemicals in host organisms, where
a host organism is transformed with one of the expression cassettes or
vectors described above, and this expression cassette comprises one or
more structural genes which code for the desired fine chemical or
catalyze the biosynthesis of the desired fine chemical, the transformed
host organism is cultured, and the desired fine chemical is isolated from
the culture medium. This process is widely applicable to fine chemicals
such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and
synthetic flavorings, aromatizing substances and colorants. The
production of tocopherols and tocotrienols, and of carotenoids is
particularly preferred. The culturing of the transformed host organisms,
and the isolation from the host organisms or from the culture medium
takes place by means of processes known to the skilled worker. The
production of pharmaceuticals such as, for example, antibodies or
vaccines is described in Hood E E, Jilka J M (1999). Curr Opin Biotechnol
10(4):382-6; Ma J K, Vine N D (1999). Curr Top Microbiol Immunol
236:275-92.

[0117] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure content
and the disclosure content specifically mentioned in this specification.

[0127] FIG. 10: The expression cassette of both GUS and DsRed reporter
genes driven by the ZmNP27 promoter in bi-directions in the construct,
RHF 175.

[0128] FIG. 11: Sequence of vector RHF175 (SEQ ID NO: 11).

[0129] FIG. 12: GUS expression in different tissues at different
developmental stages driven by ZmNP27 in forward direction in transgenic
maize with RLN88.

[0130] FIG. 13: Bi-directional function of the pZmNP27. The expression of
DsRed gene was controlled by the pZmNP27 in reverse direction. The
expression of GUS expression was controlled by pZmNP27 in forward
direction in transgenic maize with RHF175.

[0131] FIG. 14: Sequence of pZmNP18 (SEQ ID NO: 2).

[0132] FIG. 15: Sequence of pZmNP27-mini (SEQ ID NO: 3).

[0133] FIG. 16: GUS expression in different tissues at different
developmental stages driven by pZmNP18 in transgenic maize with RLN87.

[0134] FIG. 17: GUS expression in different tissues at different
developmental stages driven by pZmNP27-mini in transgenic maize with
RHF178.

EXAMPLES

[0135] The invention will now be illustrated by the following Examples
which are not intended, whatsoever, to limit the scope of this
application.

Example 1

Identification of the Maize Ortholog of NP27

[0136] In an expression profiling analysis using Affymetrix GeneChip®
Wheat Genome Arrays, the wheat chip consensus sequence TaAffx.115437.1.A1
showed constitutive expression. When the sequence of TaAffx.115437.1.A1
was aligned with the sequences of the Affymetrix maize chip, a maize chip
consensus sequence, Zm.348.2.A1_a_at was identified as an ortholog of
TaAffx.115437.1.A1 with 78% nucleotide sequence identity in the first 290
nucleotides of the TaAffx.115437.1.A1. The sequences of
TaAffx.115437.1.A1 and Zm.348.2.A1_a_at are shown in FIG. 1.

[0137] Total RNA isolated from immature embryo, leaf, young ear, and
kernel was used for this Affymetrix GeneChip® Maize Genome Array
analysis. A total of 36 arrays were hybridized. The results indicated
that Zm.348.2.A1_a_at expressed constitutively in all tested tissues
(FIG. 2).

[0138] Quantitative reverse transcriptase-polymerase chain reaction
(qRT-PCR) was performed to determine the expression levels of
Zm.348.2.A1_a_at in various types of tissues. The sequence of
Zm.348.2.A1_a_at was Blasted against the BASF Plant Science proprietary
sequence database. One maize EST ZM03MC02483--60578324 (745 bp) was
identified as a member of the gene family of Zm.348.2.A1_a_at. The
sequence of ZM03MC02483--60578324 is shown in FIG. 3.

[0139] Primers for qRT-PCR were designed based on the sequence of
ZM03MC02483--60578324 using VNTI. Two sets of primers were used for
PCR amplification. The sequences of primers are in Table 1. The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene served as a control
for normalization.

[0140] qRT-PCR was performed using SuperScript III Reverse Transcriptase
(Invitrogen, Carlsbad, Calif., USA) and SYBR Green QPCR Master Mix
(Eurogentec, San Diego, Calif., USA) in an ABI Prism 7000 sequence
detection system. cDNA was synthesized using 2-3 quadratureg of total
RNA and 1 μL reverse transcriptase in a 20 quadratureL volume. The
cDNA was diluted to a range of concentrations (15-20 ng/quadratureL).
Thirty to forty ng of cDNA was used for quantitative PCR (qPCR) in a 30
quadratureL volume with SYBR Green QPCR Master Mix following the
manufacturer's instruction. The thermocycling conditions were as follows:
incubate at 50° C. for 2 minutes, denature at 95° C. for 10
minutes, and run 40 cycles at 95° C. for 15 seconds and 60°
C. for 1 minute for amplification. After the final cycle of the
amplification, the dissociation curve analysis was carried out to verify
that the amplification occurred specifically and no primer dimer was
produced during the amplification process. The housekeeping gene
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, primer sequences in
Table 1) was used as an endogenous reference gene to normalize the
calculation using Comparative Ct (Cycle of threshold) value method. The
ΔCT value was obtained by subtracting the Ct value of GAPDH
gene from the Ct value of the candidate gene
(ZM03MC02483--60578324). The relative transcription quantity
(expression level) of the candidate gene was given by 2.sup.-ΔCT.
The qRT-PCR results were summarized in FIG. 4. Both primer sets gave the
similar expression patterns that are validated to the expression patterns
obtained from the Affymetrix GeneChip® Maize Genome Array analysis
shown in FIG. 2.

Example 4

Annotation of the Zm.348.2.A1_a_at Sequence

[0141] The coding sequence corresponding to the Zm.348.2.A1_a_at gene was
annotated based on the in silico results obtained from both BlastX of EST
ZM03MC02483--60578324 sequence against GenBank protein database (nr)
and result from VNTI translation program. The EST
ZM03MC02483--60578324 encodes a 60S acidic ribosomal protein P3
(GenBank Accession: O24413/RLA3_Maize) gene in maize. The top 15
homologous of the BlastX results are presented in table 2.

[0142] Sequence upstream of the start codon of the 60S acidic ribosomal
protein P3 gene was defined as the promoter. To identify this predicted
promoter region, the sequence of EST ZM03MC02483--60578324 was
mapped to the BASF Plant Science proprietary genomic DNA sequence
database. One maize genomic DNA sequence, ZmGSStuc11-12-04.271010.1 (880
bp) was identified. This 880 bp sequence harboured a part of the EST
ZM03MC02483--60578324 and contained partial coding sequence (CDS) of
the gene and 666 bp sequence upstream of the start codon (FIG. 6). The 5'
UTR (81 bp) was determined by the 5'RACE (Rapid Amplification of 5'
Complementary DNA Ends) and is indicated in bold and italic letters in
FIG. 6. The putative TATA signal sequence is indicated in underlined bold
letters (FIG. 6).

Example 6

Isolation of the Promoter Region by PCR Amplification

[0143] PCR was carried out using the sequence specific forward primer
GGCATGTATGGTGGAATTAT (SEQ ID NO: 18) and reverse primer
GTCGCTTGTTCCCTGCGTGC (SEQ ID NO: 19) to isolate the promoter region. A
fragment of 651 bp was amplified from maize genomic DNA. This promoter
region was named promoter ZmNP27 (pZmNP27). Sequence of pZmNP27 was shown
in FIG. 7.

Example 7

PLACE Analysis and Prediction of Bi-directional Function of the Promoter
ZmNP27

[0144] Cis-acting motifs in the 651 by ZmNP27 promoter region were
identified using PLACE (a database of Plant Cis-acting Regulatory DNA
elements) via Genomatix. The results were listed in Table 3. A putative
TATA box is located between the nucleotide (nt) sequence number 335 and
341 in the forward strand. Two putative TATA boxes are located between
the nucleotide (nt) sequence number 17 and 23 as well as 25 and 32 in the
reverse strand and two CCAAT boxes are located between the nucleotide
(nt) sequence number 84 and 88 as well as 108 and 112 in the reverse
strand. The results of this in silico analysis indicated that the pZmNP27
might function as a bi-directional promoter.

Binary Vector Construction for Maize Transformation to Identify the
Function of pZmNP27 in Forward Direction

[0145] The 651 by promoter fragment amplified by PCR was cloned into
pENTR® 5'-TOPO TA Cloning vector (Invitrogen, Carlsbad, Calif., USA).
A BASF Plant Science proprietary intron-mediated enhancement (IME)-intron
(BPSI.1) was inserted into the restriction enzyme BsrGl site that is 24
by downstream of the 3' end of the ZmNP27. The resulting vector was used
as a Gateway entry vector in order to produce the final binary vector RLN
88 that has pZmNP27::BPSI.1::GUS::t-NOS cassette for maize transformation
(FIG. 8) to characterize the function of pZmNP27 in the forward
direction. Sequence of the binary vector RLN 88 is shown in FIG. 9.

Example 9

Binary Vector Construction for Maize Transformation to Identify the
Function of pZmNP27 in Both Forward and Reverse Directions

[0146] To determine if the pZmNP27 functions bi-directionally, another
binary vector, RHF175 was constructed. The GUS reporter gene in
combination with the NOS terminator (GUS::NOS) was fused downstream of
BPSI.1 intron, which became a construct named RLN88. The GUS gene
expression was controlled by the pZmNP27 in forward direction. RLN88 also
contains a plant selectable marker cassette between LB and the GUS
reporter gene cassette. The second reporter gene, DsRed, in combination
with the NOS terminator (DsRed::NOS) was fused upstream of the 5'end of
pZmNP27 in RLN88. The expression of this DsRed gene was controlled by the
pZmNP27 in reverse direction. The tesulting construct was named RHF175.
The reporter gene cassette in RHF175 is structured as follows:
t-NOS::DsRed::pZmNP27::BPSI.1::GUS::t-NOS (FIG. 10). The sequence of
RHF175 is shown in FIG. 11.

Example 10

Promoter Characterization in Transgenic Maize with RLN 88

[0147] Expression patterns and levels driven by the ZmNP27 promoter were
measured using GUS histochemical analysis following the protocol in the
art (Jefferson 1987). Maize transformation was conducted using an
Agrobacterium-mediated transformation system. Ten and five single copy
events for T0 and T1 plants were chosen for the promoter analysis. GUS
expression was measured at various developmental stages:

[0152] 5) Ear or Kernels at 5, 10, 15, 20, and 25 days after pollination
(DAP)

[0153] The results indicated that forward direction of ZmNP27 of RHF88
functioned constitutively with preferable expression in whole seeds and
stem (FIG. 12).

Example 11

Promoter Characterization in Transgenic Maize with RHF175

[0154] Expression patterns and levels driven by the ZmNP27 promoter in
both directions were measured using GUS histochemical analysis for the
GUS reporter as stated above and using fluorescence scanner Typhoon 9400
for DsRed reporter expression. The tissue types and developmental stages
were the same as listed above.

[0155] The pZmNP27 in reverse direction expressed DsRed gene in leaf and
root but not in Seed (FIG. 13). The pZmNP27 in reverse direction
expressed DsRed gene in leaf and root but not in Seed (FIG. 13).

Example 12

Deletion Experiment of Promoter ZmNP27 to Identify the Key Regions for
Function

[0156] Two deletions were made to identify the key regions for the
promoter function:

[0157] The 159 bp fragment from the 5' end of pZMNP27 was deleted. The
remaining 492 by of the promoter region including the 5' UTR (FIG. 145)
was named pZmNP18. The pZmNP27 in RLN88 was replaced with pZmNP18, which
became a construct named RLN87.

[0158] The 380 bp from the 5' end of pZMNP27 was deleted. The remaining
271 by promoter region including the 5'UTR (FIG. 15) was named
pZmNP27-mini. The pZmNP27 in RLN88 was replaced with pZmNP27-mini, which
became a construct named RHF178.

[0159] Both pZmNP18 and pZmNP27-mini functioned very similar to the full
length of forward pZmNP27 in maize. The expression results in transgenic
plant with RLN87 and RHF178 are shown in FIG. 16 and FIG. 17,
respectively.